USPTO Patents Application 10002961

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Title of the Invention
A System and Method for Investigating the Effect
of Chemical and Other Factors on Cell Movement

Reference to Related Applications
[0001] This application claims benefit under Title 35, U.S.C. § 1 19(e), of

United States Application Serial No. 60/243,450, filed October 26, 2000.

Field of the Invention

[0002] The present invention relates, in general, to novel methods for

measuring cell movement and to methods for assessing the impact of a variety
of factors on the ability of cells to move. In particular, the present invention
relates to systems and methods for measuring cell movement and the effect of
various chemical species on that phenomenon by monitoring one or more
electrical parameters of sensing electrodes that are sensitive to the interaction of
cells with the electrode surface.

Background of the Invention

[0003] Cells move from place to place in multicellular organisms for a

variety of reasons. For example, cell movement occurs during organogenesis,
movement is essential to inflammatory immune responses, and movement of
neoplastic cells permits metastasis to secondary sites. This movement can arise
from the intrinsic characteristics of the cell, or it can be initiated, enhanced or
otherwise affected by the presence of external chemical stimuli. Stimuli can be
divided into two classes: those that stimulate cell movement without a specific
directional aspect (chemokinesis), and those stimuli that enhance directional cell
movement according to the location of external cues (chemotaxis).

[0004] Cell movement is a critical component of both normal immune

function and the dysfunctional immune responses associated with certain
disease conditions such as asthma, chronic inflammation and autoimmune
disease. Often this movement is characterized as chemotactic, and is initiated in
response to the presence of one or more of a set of chemoattractants. These
chemoattractant species (e.g., chemokines and activated components of the
complement cascade) can be produced by the body in response to a variety of
stimuli. Additionally, cells of the immune system are capable of rapid and
vigorous responses to stimuli provided by infectious microorganisms (e.g. , /"-met-

leu-phe and other formylated peptides that are products of bacterial protein
degradation). In the context of an infection or other inflammation, these signals
cause the influx of cells (notably macrophages and neutrophils) at the site of
inflammation. The common mechanism of action for these signals is to engage
specific receptors on the surface of the cell. Following ligand/receptor
engagement, one or more signal transduction cascades are initiated, and the cell
responds by specific activation of genes and the movement of the cell along the
gradient. Still unknown is the means by which these cells sense the gradient,
and the actual mechanisms by which they move through the cellular
environment to arrive at the source of the chemoattractant.

[0005] From a practical standpoint, studies that identify new chemokines

and other attractants, that characterize the signal transduction cascade and the
differentiation of the responding cell, as well as studies that characterize the
environment in which movement occurs, will each provide potential avenues of
therapeutic manipulation. Assays that measure cellular movement in response
to a chemotactic gradient offer the ability to assess individual elements along the
length of the path from initiation of the response to the cellular accomplishment
of the movement.

[0006] Quantitative and qualitative measurement of cell movement can be

important to the characterization of biological responses, such as those
mentioned above, as well as to many others. The rational design of therapeutic
strategies for clinical intervention in these systems can theoretically depend
upon manipulation of cellular motility: increasing it when a more robust response
is desired, and diminishing the influx of cells to reduce their contributions to the
response. For example, pharmacological manipulations of cell accumulation in
the airways has been found to be an effective treatment for some forms of
asthma, and interference with cellular movement through the vascular epithelia
can diminish some the inflammatory damage associated with ischemia/re-
perfusion injury. Vigorous research efforts are currently underway in many
biotechnology and pharmaceutical laboratories to discover novel therapeutics
with the capacity to affect cell movement. For example, it is understood in the
art that a potential therapeutic approach is to use inhibitors of signal
transduction to manipulate chemotactic responsiveness, and many investigations
are currently under way to assess the viability of such an approach in the

treatment of a number of disease conditions. Essential to these investigations is
the capacity to make qualitative and quantitative observations of cell movement
in response to chemotactic stimuli, as well as mediation of that response by
inhibitors or enhancers of chemotactic response.

[0007] In the prior art, measurement of cell movement directed by

chemotactic agents has been accomplished in several ways. A "small-
population" assay can optically measure the movement of cells in an initial
localized deposit of these cells in a chemotactic gradient that exists in proximity
to the cells. Variations of the Boyden chamber assay (Boyden, S., Journal of
Experimental Medicine 115: 453 (1953)) are currently the most commonly used.
In these assays, the cells are placed on a microporous membrane over a source
of chemotactic agent. As the cells detect the higher concentrations of
chemotactic agent that diffuse from the source, they migrate through the
membrane to its underside. Migrating cells are usually statically detected by
manual and optically aided methods on the reverse side of the membrane after
staining. The quantity of responding cells is usually determined as an endpoint
assay at a predetermined time-point. Thus, assays of this sort are usually
capable of a semi-quantitative measure of the number of cells from an initial cell
population that travel across a membrane in response to a perceived gradient of
a chemoattractive agent. An advantage of this type of technique is the ability to
perform many simultaneous assays, as multi-well plates in a two-dimensional
array may be effectively utilized. However, a major limitation of a Boyden-type
assay is that the chemical gradient sensed by the cells is very steep and
dissipates rapidly. Essentially, there is a high concentration of chemoattractant
on one side of the separating membrane and none on the other. In addition, it is
also difficult to visualize the movement of cells through the membrane in this
chemotactic environment. Finally, quantitation of the number of cells to move in
response to the chemotactic stimulus is limited by traditional cell-counting
methodologies and other errors inherent in the system such as the loss of
migrated cells from the underside of the membrane where counting occurs.

[0008] Another technique used to measure chemotaxis is to track cell

movement by video microscopy in a Zigmond or Dunn chamber. In these
assays, the movement of cells is recorded as they respond to an aqueous
gradient of chemoattractant formed between two closely spaced glass surfaces.

This assay suffers from serous drawbacks in that it is more difficult to set up,
only a small number of cells can be analyzed at one time, and the assay cannot
be easily multiplexed.

[0009] The under-agarose chemotactic assay (Nelson, R.D., et al. Journal

of Immunology 115: 1650-1656 (1975)) provides a different approach from that
offered in other, Boyden-type assays. In the under-agarose assay, a planar layer
of agarose gel is cast in a cell culture dish. Multiple wells are cut in the agarose
layer with a device such as a stainless steel punch. In a typical assay, multiple
sets of three wells are punched in a linear array. In the middle well of the three-
well set, a portion of cell suspension is added to the well. In one of the
adjoining wells, a solution of a chemotactic agent is added. In the third well, a
suitable control solution is added. The assembly is allowed to incubate at an
appropriate temperature for a pre-determined period of time after which the cells
are fixed with the agarose layer in place by the addition of suitable fixing agents
such as absolute methanol. After fixation, the gel layer is removed and the
plates stained. The migration patterns of the cells are observed optically and
measurements taken of individual cells along paths toward the chemoattractant
well and compared with the movement of cells toward the control well.

[0010] This type of assay provides a significantly different type of cell

environment than that utilized in a Boyden-type assay. First, the cells under
investigation move while surrounded by the underlying substrate (glass culture
dish) and the overlying agarose layer. Second, the chemotactic gradient is
stabilized by the agarose allowing the gradient to be established over a larger
volume, and for a longer period of time. As indicated above, the under-agarose
assay measures the distance cells move in a specified period of time as an
indication of a chemotactic response. This assay has the advantage that a
single endpoint need not be evaluated since the cells gradually spread away from
the starting well. The disadvantage is that in running many parallel assays, each
would have to be evaluated microscopically at many time points to get an
estimate of the extent of movement in each assay. Furthermore, the nature of
the measurements obtained with this assay render it very difficult to quantify the
rate at which cells move in response to chemotactic stimuli.

[0011] In the interest of obtaining information on a totally different type

of cell motion, Giaever and Keese have developed an electrochemical-based

system for assessing cell motility, as disclosed in United States Patent No.
5,187,096, the disclosure of which is hereby incorporated in its entirety. A
commercialization of this system, known as the Electric Cell Impedance Sensing
("ECIS") system has been developed and is sold by Applied Biophysics, Inc.
(Troy, NY).

[0012] In the ECIS assay system, two electrodes are lithographed onto

the surface of a lexan slide and positioned within a chamber that holds aqueous
media. Cells in this media can attach to a sensing electrode and to the
surrounding surface of the slide. A 1 volt a.c. current passes through the
culture media that functions as an electrolyte, and a lock-in amplifier measures
current flow through this circuit. This measurement provides data on the initial
resistance of the system and, more importantly, any changes to current flow on
the electrode that occur over time. Due to the relatively small size of the
electrode, resistance at the sensing electrode predominates in the system. Any
activity that affects the adherence of cells to the electrodes will alter the
measured electrical resistance in the system. For example, increasing the
tightness of association of cells with the surface of the electrode by coating it
with extracellular matrix proteins increases the resistance of the electrical circuit.
Lipopolysaccharide (LPS) activates macrophages to spread and cover a larger
amount of the target electrode and thus also increase the resistance measured at
the target electrode. In contrast, toxicants that damage cells will act to reduce
the resistance of the circuit. The degree of or changes in cell motility will also
be reflected by changes in the measured electrode resistance as the extent of
interaction between the cells and the electrode surface changes.

[0013] FIG. 1 illustrates a typical prior art ECIS configuration with a side

view (not to scale) of cells 54 sitting on the sensing electrode 10 in a culture
well 50. The electrodes comprise gold electrodes fabricated on plastic substrata
58. Culture media 55 is used as the electrolyte. In a typical ECIS application, a
constant AC current of 1 microampere at 4 kHz is maintained between the
sensing electrode 10 and a large counter electrode 40, while the voltage is
monitored with a lock-in amplifier 52. Voltage and phase data are stored and
processed with a microprocessor 60. Normally, these data are converted to
resistance or capacitance by treating the cell-electrode system as a series RC
circuit. The same microprocessor controls the output of the amplifier and

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switches the measurement to different sensing electrodes in the course of an
experiment with a multi-cell array.

[0014] In an ECIS system, the relative sizes of the sensing and counter

electrodes can be significant. With larger sensing electrodes, cell-related
resistance signals become difficult to detect. This is a consequence of bulk
solution resistances that tend to swamp out the contribution to total resistance
from the sensing electrode. "When electrodes have a surface area of
approximately 10 3 cm 2 or less, the impedance of the electrode-electrolyte
interface at 4 kHz predominates, and in this situation, changes in resistance due
to interaction of the cells with the electrode surface are clearly revealed.

[0015] In a typical assay, cells seeded into an ECIS well settle to the

bottom of the well, attach to the surface of the sensing electrode 10 that is
fabricated on the bottom surface of the well, and individual cells spread radially.
The number of cells on the well, the intimacy of contact, the degree of
spreading, and the activity (motility) of the cells all contribute to the level of
resistance imparted by the cells to the circuit. A single electrode can be
monitored as often as four times per second with currently available hardware in
the commercial embodiment of the ECIS system. The intimacy of cell contact
with the electrode can be modified by pre-incubation of the electrode with
different extracellular matrix proteins and this can result in different levels of
resistance imparted by the cells to the system. Moreover, the intimacy of
contact can be modified by exposing the cells to agents that alter the viability,
signal transduction, or membrane integrity of the cell.

[0016] As disclosed in U.S. Patent No. 5,187,096, cited above, the ECIS

system is directed toward investigations of cellular phenomena that are only
remotely implicated in the type of cell movement associated with chemotactic or
chemokinetic behavior. As such, its utility, although specialized, does not
extend in its conventional applications to investigations into the mechanism of
translational cell movement, or the influence of chemical agents on that motion.

[0017] Consequently, there exists a need in the art for an assay system

directed toward translational cell movement that is capable of rapid, automated
and multiplexed analysis of cell movement and factors capable of affecting such
movement. In a unique combination of the traditional under-agarose cell assay

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with the specific capabilities of an ECIS system, the present inventors have
developed a system and methods for investigation of phenomena associated
with cell movement that possesses these desirable characteristics, and
addresses the majority of the shortcomings associated with prior art techniques.
Specific embodiments of these systems and methods are detailed below.

Summary of the Invention
[0018] In a first embodiment, the present invention provides a system for

monitoring the effect of extracellular chemical stimuli on the translational motion
of cells, the system comprising: (a) an array of one or more cell containment
volumes; (b) an array of one or more chemical agent volumes interspersed
among the array of one or more cell containment volumes; (c) one or more
substantially planar sensing electrodes distributed within the arrays of cell
containment volumes and chemical agent volumes so that at least one of the
sensing electrodes is between one cell containment volume and one chemical
agent volume, wherein the one or more sensing electrodes is operatively coupled
to a sensing device capable of measuring an electrical parameter of the sensing
electrode; (d) at least one counter electrode in electrical connection with the one
or more sensing electrodes; and (e) a biocompatible chemical gradient stabilizing
medium in simultaneous diffusional contact with the arrays of cell containment
volumes and chemical agent volumes. In this embodiment of the present
invention, the at least one counter electrode and the one or more sensing
electrodes are connected in series.

[0019] In addition, the present invention contemplates a system wherein

the at least one counter electrode and the one or more sensing electrodes are
connected in parallel. Alternatively, the at least one counter electrode and the
one or more sensing electrodes are connected in series. Also, the system of the
invention can further comprise a reference electrode in electrical connection to
the at least one counter electrode and the one or more sensing electrodes. The
system of the invention also contemplates that the measured electrical
parameter of the sensing electrode is impedance, or resistance, or capacitance.
[0020] As exemplified in this embodiment, the system of the invention

contemplates a chemical gradient stabilizing medium that is in a planar geometry
overlying the arrays of cell containment volumes and chemical agent volumes.

Preferably, the chemical gradient stabilizing medium is an agarose gel.
Furthermore, preferentially, the geometry of the sensing electrode is
substantially circular. Alternatively, the geometry of the sensing electrode can
be substantially rectangular. It is also possible that the geometry of the sensing
electrode is semi-circular. Preferably, the surface area of each of the one or
more sensing electrodes is from about 0.5 x 10 2 mm 2 to about 10 x 10 2 mm 2 .

[0021] In a particularly preferred embodiment, the system of the present

invention comprises a sensing device that is operatively coupled to a
microprocessor. This microprocessor can be connected to an output display
device capable of displaying the electrical parameter values measured at the one
or more sensing electrodes. Preferably, the output display device is a cathode
ray tube (CRT), or alternatively, a hard copy device such as plotter or printer.
More preferably, the microprocessor is under the control of a software program
executable on the microprocessor.

[0022] In yet another embodiment, the present invention provides a

method for monitoring the translational motion of cells in response to
extracellular chemical stimuli, the method comprising the steps of (a) placing a
population of one or more cells in a biocompatible medium into a cell
containment volume; (b) placing a chemical agent in a biocompatible medium
into a chemical agent volume in diffusional contact with a biocompatible
chemical gradient stabilizing medium; and (c) monitoring changes in an electrical
parameter of one or more substantially planar sensing electrodes interposed
between the cell containment volume and the chemical agent volume and in
electrical connection with a counter electrode, wherein the changes in electrical
parameter of the one or more sensing electrodes arise substantially from contact
of one or more cells from the cell population with a surface of one or more of
the sensing electrodes, and wherein the one or more cells have diffused to the
surface of one or more of the sensing electrodes from the cell containment
volume under the influence of a chemical gradient of the chemical agent in the
chemical gradient stabilizing medium.

[0023] According to the present embodiment of the claimed invention, the

measured electrical parameter is impedance. Alternatively, the measured
electrical parameter is resistance or capacitance. In another aspect of this
embodiment, the translational movement of the one or more cells is directionally

focused. Alternatively, the translational movement of the one or more cells is
not directionally focused. In yet another aspect of this embodiment, there is
additionally interposed between the cell containment volume and the one or
more sensing electrodes one or more barriers to translational motion of the cells.
The present invention contemplates that the barrier is physical in nature.
Alternatively, the barrier may be chemical in nature. In an alternative
configuration of this embodiment of the claimed invention, the sensing electrode
and the counter electrode are in electrical connection with a reference electrode.

[0024] In the practice of the present invention, the one or more cells are

exposed to two or more independent chemical gradients from different chemical
agents. In this aspect, the independent chemical gradients are physically
overlapping. Alternatively, the independent chemical gradients are not physically
overlapping.

[0025] According to this embodiment of the present invention, the cells of

the cell population are selected from the group consisting of D. discoideum,
bone marrow cells from BALB/c mice, M1 cells, U937 cells, and other motile
eukaryotic cells from both tissue culture and from living animals.

[0026] In addition, in the practice of the claimed invention, the chemical

agent may be selected from the group consisting of folic acid, guinea pig serum,
activated complement, bacterial peptides, and mammalian chemokines.

[0027] The present embodiment also contemplates that the chemical

agent volume is the biocompatible chemical gradient stabilizing medium.

[0028] In still another embodiment, the claimed invention provides a

method for determining the impact of a test substance on the ability of a
chemical agent to affect the translational movement of cells, the method
comprising the steps of (a) placing a population of one or more cells in a
biocompatible medium into a cell containment volume; (b) placing a chemical
agent in a biocompatible medium into a chemical agent volume in diffusional
contact with a biocompatible chemical gradient stabilizing medium; (c) exposing
one or more cells of the population to a test substance; (d) monitoring one or
more electrical parameters measured on a substantially planar sensing electrode
positioned between the cell containment volume and the chemical agent volume,
wherein the changes in impedance on the sensing electrode arise substantially

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from contact of one or more cells from the cell population with a surface of the
sensing electrode, and wherein the one or more cells have diffused to the
surface of the sensing electrode from the cell containment volume under the
influence of a chemical gradient of the chemical agent in the chemical gradient
stabilizing medium between the cell containment volume and the chemical agent
volume; and (e) comparing the one or more electrical parameters measured in
step (d) with electrical parameter measurements taken for one or more cells from
the population that have not been exposed to the test substance.

[0029] As practiced, the method of the present invention contemplates

that the measured electrical parameter is impedance. Alternatively, the
measured electrical parameter is resistance or capacitance.

[0030] In addition, the method of the claimed invention further

contemplates exposing the cells to a second test substance and comparing the
resulting measured electrical parameter readings with corresponding electrical
measurements taken for one or more cells from the population that have been
exposed to the first test substance but not the second test substance.

[0031] In one aspect of this embodiment of the claimed invention, the

translational movement of the one or more cells is directionally focused.
Alternatively, the translational movement of the one or more cells is not
directionally focused. In yet another aspect, there is additionally interposed
between the cell containment volume and the one or more sensing electrodes
one or more barriers to translational motion of the cells. The one or more
barriers may be physical in nature and/or chemical in nature.

[0032] In an alternative embodiment, the present invention provides a

system for the non-optical imaging of translational cell movement comprising (a)
one or more cell containment volumes; (b) one or more chemical agent volumes;
(c) a plurality of sensing electrodes interposed between the cell containment
volumes and the chemical agent volumes, wherein each of the plurality of
sensing electrodes is operatively coupled to a sensing device capable of
measuring an electrical parameter of the sensing electrode; (d) at least one
counter electrode in electrical connection with the array of sensing electrodes;
and (e) a biocompatible chemical gradient stabilizing medium in simultaneous
diffusional contact with the cell containment volumes and the chemical agent

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volumes. Preferably, the plurality of sensing electrodes are arranged in an
orderly, two-dimensional array. In this embodiment, the dimensions of individual
sensing electrodes is of an order that is not much larger than the dimensions of a
typical cell such that the electrode surface is large enough to hold only one cell
at a time. As configured, this embodiment of the invention comprises an array
of at least 100 sensing electrodes. Preferably, the array comprises at least
1000 sensing electrodes. More preferably, at least 2500 sensing electrodes.

[0033] In this embodiment the electrical parameter measured at the

sensing electrode is impedance. Alternatively, the electrical parameter is
resistance or capacitance. This embodiment of the invention also contemplates
the further inclusion of a reference electrode in electrical connection to the at
least one counter electrode and the array of sensing electrodes. In addition, the
present invention provides that the chemical gradient stabilizing medium is in a
planar geometry overlying the arrays of cell containment volumes and chemical
agent volumes. Preferably, the chemical gradient stabilizing medium is an
agarose gel.

[0034] In a particularly preferred embodiment, the system of the present

invention comprises a sensing device that is operatively coupled to a
microprocessor. This microprocessor can be connected to an output display
device capable of displaying the electrical parameter values measured at the one
or more sensing electrodes. Preferably, the output display device is a cathode
ray tube (CRT), or alternatively, a hard copy device such as plotter or printer.
More preferably, the microprocessor is under the control of a software program
executable on the microprocessor.

Brief Description of the Drawings

[0035] FIG. 1 is a schematic representation of a prior art ECIS system for

the electrical measurement of certain types of cell activity indicative of cell
motility.

[0036] FIG. 2, in three panels, A - C, provides a schematic representation,

in three views, of the cell movement assay system of the present invention.

[0037] FIG. 3, in panels A - D, is an illustration of alternative electrode

geometries for the cell movement assay system of the present invention.

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[0038] FIG. 4 is a plot of normalized resistance as a function of time

illustrating the movement of Dictyostelium discoidium cells in response to a folic
acid gradient.

[0039] FIG. 5, in four panels, A - D, is a series of video images of the

sensing electrode for which resistance data was plotted in FIG. 4 at each of the
four time points specifically illustrated in FIG. 4.

[0040] FIG. 6 is a plot of normalized resistance as a function of the

number of cells detected on the surface of a sensing electrode of the cell
movement assay system of the present invention.

[0041] FIG. 7 is a plot of normalized resistance measured at the sensing

electrode of the cell movement assay system of the present invention in a series
of experiments with an increasing concentration of folic acid as the
chemoattractant species.

[0042] FIG. 8 is a plot of normalized resistance measured at the sensing

electrode of the cell movement assay system of the present invention where cell
movement was in response to a uniform concentration of chemoattractant in the
gradient stabilizing medium.

[0043] FIG. 9 is a plot of normalized resistance measured at the sensing

electrode of the cell movement assay system of the present invention in a series
of experiments with mutant Dictyostelium discoidium cells lacking the gene for
the myosin II heavy chain.

[0044] FIG. 10 is a plot of normalized resistance measured at the sensing

electrode of the cell movement assay system of the present invention in a series
of experiments illustrating the effect of exposure to increasing concentrations of
cisplatin on the chemotactic movement of cells in response to folic acid as a
chemoattractant species.

[0045] FIG. 1 1 is a plot of normalized resistance measured at the sensing

electrode of the cell movement assay system of the present invention in a
system configuration comprising a third (reference) electrode.

[0046] FIG. 12, in four panels, A - D, is a series of video images of the

sensing electrode for which resistance data was plotted in FIG. 1 1 at each of the
four time points specifically illustrated in FIG. 1 1 .

Detailed Description of the Invention

[0047] In a first embodiment, the present invention provides a system for

monitoring the effect of extracellular chemical stimuli on the translational motion
of cells, the system comprising: (a) an array of one or more cell containment
volumes; (b) an array of one or more chemical agent volumes interspersed
among the array of one or more cell containment volumes; (c) one or more
substantially planar sensing electrodes distributed within the arrays of cell
containment volumes and chemical agent volumes so that at least one of the
sensing electrodes is between one cell containment volume and one chemical
agent volume, wherein the one or more sensing electrodes is operatively coupled
to a sensing device capable of measuring an electrical parameter of the sensing
electrode; (d) at least one counter electrode in electrical connection with the one
or more sensing electrodes; and (e) a biocompatible chemical gradient stabilizing
medium in simultaneous diffusional contact with the arrays of cell containment
volumes and chemical agent volumes. In this embodiment of the present
invention, as illustrated in panel A of FIG. 1, the at least one counter electrodes
40 and the one or more sensing electrodes 10 are connected in series although,
alternatively, the present invention contemplates a system wherein the at least
one counter electrode and the one or more sensing electrodes are connected in
parallel. Also, the system of the invention can further comprise a reference
electrode in electrical connection to the at least one counter electrode and the
one or more sensing electrodes. The system of the invention also contemplates
that the measured electrical parameter of the sensing electrode is impedance, or
resistance, or capacitance.

[0048] In contrast to the present invention, prior art studies of cell

motility are usually conducted in a laboratory by observing cells crawling on a
glass coverslip in liquid media. Under these conditions, there is little to resist the
movement of the cells except their own adhesion to the substrate. However,
cells in natural environments, such as amoebae moving in the soil or neutrophils
extravasating through the endothelium of a capillary, are presumed to move
under more restrictive conditions. In addition, movement in three-dimensional
environments has the added complexity that cells do not have a clearly defined
dorsal and ventral surface since they can interact with the substrate on all sides.

The molecular mechanisms underlying motility in three-dimensional environments
are as yet poorly defined.

[0049] Using the system of the present invention, it is possible to

establish a stable chemotactic gradient in which cell responses can be measured
in real time. The present system is sufficiently sensitive to detect the arrival of a
single cell at the surface of the sensing electrode. Moreover, the time of arrival
of migrating cells at the target electrode is relatively uniform, and enables the
identification of a wave-like behavior in the movement of these cells. The
Examples presented below demonstrate that the system of the present invention
can be used to characterize chemoattractants, soluble antagonists of chemotaxis
or novel mutants affecting chemotaxis.

[0050] The claimed system of the present invention builds upon a

standard configuration of the ECIS system of Giaever and Keese to provide
unique capabilities that facilitate the automated monitoring of cell population
movement over time. To begin with, the system of the present invention
provides a chemical gradient stabilizing medium in the form of an agarose layer
64 that covers and is in diffusional contact with the individual wells of a typical
multi-chamber sample system as illustrated in Panel C of FIG. 2. This enables
the establishment of a chemotactic gradient between the one or more chemical
agent volumes 66 loaded with chemoattractant and the cell containment
volumes 68 in which cells are initially loaded.

[0051] The agarose layer 64 that serves as the chemical gradient

stabilizing medium permits the establishment of the necessary chemical gradient
as chemoattractant species begin to diffuse out of the chemical agent volumes
66. Due to the unique, art-recognized physical and chemical properties of a
medium such as agarose, the resulting chemical gradient that is sensed by the
cells in the cell containment volume comprises a greater volume and persists for
a much longer time than the type of gradient that exists in the prior art Boyden-
type chemotactic assay. As will be appreciated by one of skill in the art,
alternative materials for selection of the gradient stabilizing medium are readily
available. Such materials must be biocompatible with the cellular species under
investigation; must provide a solution-like environment in which likely
chemoattractant species are soluble; and must possess the necessary physical
properties to enable controlled diffusion of soluble species through the medium.

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One of skill in the art, without undue experimentation, would be fully able to
select alternative materials on the basis of such criteria.

[0052] The cells loaded into the one or more cell containment volumes

begin to move under the influence of the gradient established by diffusion of the
chemoattractant species over the substrate 58 and under the agarose layer 64 in
the direction of the gradient and interact with the one or more sensing
electrodes 10 in their path. The cells eventually reach and move across the
sensing electrode 10 located, in a preferred embodiment, between the cell
containment volume 68 and the chemical agent volumes 66, as illustrated in
panel B of FIG. 2. In one embodiment, the dimensions of the sensing electrode
are considerably greater than that of typical cells of interest, whose diameters,
on average, would be on the order of 10 pm (1 x 10 5 m). By monitoring the
changes in electrical parameters on the sensing electrode that occur over time
and the occurrence of the resulting rapid transient fluctuations in resistance, for
example, the arrival of cells at the electrode can be noted and measured.

[0053] Preferably, as is illustrated schematically in FIG. 2, the geometry

of the sensing electrode 10 is substantially circular. More preferably, the
surface area of each of the one or more sensing electrodes is from about 0.5 x
10 2 mm 2 to about 10 x 10 2 mm 2 . However, the practice of the present
invention is not constrained to a circular geometry for the one or more sensing
electrodes. As illustrated in FIG. 3, panels A - D, the sensing electrode 10, as
well as the cell containment 30 and chemical agent volumes 20, can assume a
number of alternative geometries. In addition, a negative chemotactic agent can
be placed in a second well 40 to allow the simultaneous investigation of the
effect of two different agents on cell movement. The geometry of the sensing
electrode can also be substantially rectangular, as illustrated in Panels C and D
of FIG. 3. It is also possible that the geometry of the sensing electrode is semi-
circular.

[0054] It will be appreciated that as the geometry of the sensing electrode

varies, the nature of the electrical response measured at the sensing electrode
may vary as well. As has been mentioned above, the movement of cells from a
cell containment volume of the present system under the driving force of the
chemical gradient of chemoattractant species can be detected as a psuedo-
wavefront. However, the geometries of the cell containment volumes and the

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chemical agent volumes may also influence the nature of the cell front that
reaches and is measured on the sensing electrode surface. Assuming as a first
principle that the dimensions of the cell containment and chemical agent
volumes are significantly smaller than the respective distances separating them
from the one or more sensing electrodes, it is possible to treat the cell
containment volumes and the chemical agent volumes as point sources for the
species that diffuse from them. Thus, as would be expected for a diffusional
point source, the expanding gradient of chemoattractant diffusing through the
agarose layer would present a circular front of chemical species. For the cells
moving across the substrate, it can be assumed that, in the absence of a motive
force such as a gradient of chemoattractant species, the movement of cells
would be non-directional in nature and would, over time, present a circular or
curved front as if from a point source. Deviations in the geometry of the
containment volumes would be expected to alter the nature of both the chemical
gradient through the agarose, as well as potentially the nature of the front of the
moving cells. Of course, under the influence of a chemoattractant species, even
from a small, circular source, the movement of cells would not be expected to
assume the type of form associated with random, diffusional movement.
Deviations from the expected or observed characteristics of the moving front of
cells may be capable of providing additional insight into the mechanisms of both
cell movement and cellular response to chemotactic agents, as well as to the
effects of synthetic and natural agents that alter chemotactic and chemokinetic
response.

[0055] In a particularly preferred embodiment, the system of the present

invention comprises a sensing device that is operatively coupled to a
microprocessor. The interfacing of a microprocessor driven apparatus to the
system of the present invention can greatly facilitate the collection,
transformation, analysis and display of data from the sensing electrodes. Such a
microprocessor can be connected to an output display device capable of
displaying the electrical parameter values measured at the one or more sensing
electrodes. Preferably, the output display device is a cathode ray tube (CRT), or
alternatively, a hard copy device such as plotter or printer. More preferably, the
microprocessor is under the control of a software program executable on the
microprocessor. Commands executed on the microprocessor by the software

- 17-

are used to quantify the time of arrival of cells at the sensing electrode
according to: (a) the development of a significant increase in the normalized
resistance and (b) the development of the resistance fluctuations that are
indicative of a cellular presence on the electrode. Software can be used to
automatically calculate a speed of response for the cell population according to
the time that cells first arrive on the small electrode. Software will also be able
to make comparisons between cells operating under the sole influence of
chemoattractant with cells that are exposed to chemoattractant in the presence
of inhibitors of cell movement.

[0056] In yet another embodiment, the present invention provides a

method for monitoring the translational motion of cells in response to
extracellular chemical stimuli, the method comprising the steps of (a) placing a
population of one or more cells in a biocompatible medium into a cell
containment volume; (b) placing a chemical agent in a biocompatible medium
into a chemical agent volume in diffusional contact with a biocompatible
chemical gradient stabilizing medium; and (c) monitoring changes in an electrical
parameter of one or more substantially planar sensing electrodes interposed
between the cell containment volume and the chemical agent volume and in
electrical connection with a counter electrode, wherein the changes in electrical
parameter of the one or more sensing electrodes arise substantially from contact
of one or more cells from the cell population with a surface of one or more of
the sensing electrodes, and wherein the one or more cells have diffused to the
surface of one or more of the sensing electrodes from the cell containment
volume under the influence of a chemical gradient of the chemical agent in the
chemical gradient stabilizing medium.

[0057] The present system may be used for numerous pharmacological

assays as would be readily apparent to one of ordinary skill in the art. For
example the system can be used to measure cell responses to chemoattractants
in the presence of pharmacological inhibitors of cell movement and to assess the
impact of exposure to free radicals on the ability of cells to move (sites of
infection, inflammation, and neoplastic disease often have high levels of reactive
oxygen species that may influence cell movement).

[0058] In the practice of the present invention, the one or more cells can

be exposed to two or more independent chemical gradients from different

chemical agents. In this aspect, the independent chemical gradients are
physically overlapping. Alternatively, the independent chemical gradients are not
physically overlapping. According to this embodiment of the present invention,
the cells of the cell population are selected from the group consisting of D.
discoideum , bone marrow cells from BALB/c mice, M1 cells, U937 cells, and
other motile eukaryotic cells from both tissue culture and from living animals.

[0059] In addition, in the practice of the claimed invention, the chemical

agent may be selected from the group consisting of folic acid, guinea pig serum,
activated complement, bacterial peptides, and mammalian chemokines.

[0060] In an alternative embodiment, the present embodiment

contemplates that the chemical agent volume is the biocompatible chemical
gradient stabilizing medium. Thus, a known or suspected chemoattractant can
be distributed uniformly throughout the chemical gradient stabilizing medium by
methods well known to those of skill in the art. As a result, the cells in the cell
containment volume do not sense a spreading diffusional front of
chemoattractant species. Instead, the cells are confronted initially by a uniform
chemical gradient that extends infinitely from the perspective of the cells. If the
interaction of the cells with the chemoattractant species results in a chemical
transformation of that species or a consumption of the species, the action of the
cells will begin to create a depletion zone around the cells. This cell-initiated
gradient then provides a further motive force for additional movement of the
cells in the direction of the expanding gradient, providing a unique perspective
on the factors influencing chemotactic cell movement.

[0061] Thus, in still another embodiment, the claimed invention provides a

method for determining the impact of a test substance on the ability of a
chemical agent to affect the translational movement of cells, the method
comprising the steps of (a) placing a population of one or more cells in a
biocompatible medium into a cell containment volume; (b) placing a chemical
agent in a biocompatible medium into a chemical agent volume in diffusional
contact with a biocompatible chemical gradient stabilizing medium; (c) exposing
one or more cells of the population to a test substance; (d) monitoring one or
more electrical parameters measured on a substantially planar sensing electrode
positioned between the cell containment volume and the chemical agent volume,
wherein the changes in impedance on the sensing electrode arise substantially

#

-19-

from contact of one or more cells from the cell population with a surface of the
sensing electrode, and wherein the one or more cells have diffused to the
surface of the sensing electrode from the cell containment volume under the
influence of a chemical gradient of the chemical agent in the chemical gradient
stabilizing medium between the cell containment volume and the chemical agent
volume; and (e) comparing the one or more electrical parameters measured in
step (d) with electrical parameter measurements taken for one or more cells from
the population that have not been exposed to the test substance. In addition, the
method of the claimed invention further contemplates exposing the cells to a
second test substance and comparing the resulting measured electrical
parameter readings with corresponding electrical measurements taken for one or
more cells from the population that have been exposed to the first test
substance but not the second test substance.

[0062] The analysis of cell movement in the presence of chemotactic and

chemokinetic stimuli is relevant to many different lines of basic and applied
research. One of the potential beneficial utilities of the system and method of
the present invention is for the study of normal immune processes, as well as for
the study of disease processes including chronic inflammation, autoimmune
disease, and cancer. Because the system and method of the present invention
can utilize computerized assessment of experimental results according to an
objective algorithm, and is also amenable to robotic set up and data capture, it
will provide additional utility in many pharmaceutical and biotechnology
applications, including the evaluation of anti-inflammatory drugs and in the
clinical evaluation of patient immune function. The practice of the present
invention also contemplates assessment of the movement of neoplastic cells and
drugs that alter that form of cellular movement and, thus, provides unique
assays of substances of potential therapeutic utility in the treatment of cancer.
In addition, the design of the system of the present invention is amenable to a
scaling up of the system to include larger numbers of chambers that will allow
high throughput screening of mutant cells that have alterations in their
chemotactic response, enabling the rapid identification of genes involved in
regulation of cellular movement.

[0063] In one aspect of this embodiment of the claimed invention, the

translational movement of the one or more cells is directionally focused

(chemotactic). Alternatively, the translational movement of the one or more
cells is not directionally focused (chemokinetic). In the latter instance, such
movement is typically associated with the impact of "scatter agents" on cell
movement that have been implicated in essential cellular functions associated
with a cancerous disease state. Thus, the practice of the present invention is
adaptable to investigations of non-directional cell movement associated with
tumor growth and metastasis.

[0064] In yet another aspect, there is additionally interposed between the

cell containment volume and the one or more sensing electrodes one or more
barriers to translational motion of the cells. The one or more barriers may be
physical in nature and/or chemical in nature. By observation of the interaction of
cells with these barriers and the impact of this interaction on the movement of
cells, it is possible to elucidate additional information on the mechanism of cell
movement.

[0065] In an alternative embodiment, the present invention provides a

system for the non-optical imaging of translational cell movement comprising (a)
one or more cell containment volumes; (b) one or more chemical agent volumes;
(c) a plurality of sensing electrodes interposed between the cell containment
volumes and the chemical agent volumes, wherein each of the plurality of
sensing electrodes is operatively coupled to a sensing device capable of
measuring an electrical parameter of the sensing electrode; (d) at least one
counter electrode in electrical connection with the array of sensing electrodes;
and (e) a biocompatible chemical gradient stabilizing medium in simultaneous
diffusional contact with the cell containment volumes and the chemical agent
volumes. Preferably, the plurality of sensing electrodes are arranged in an
orderly, two-dimensional array. In this embodiment, the dimensions of individual
sensing electrodes is of an order that is not much larger than the dimensions of a
typical cell such that the electrode surface is large enough to hold only one cell
at a time. As configured, this embodiment of the invention comprises an array
of at least 100 sensing electrodes. Preferably, the array comprises at least
1000 sensing electrodes. More preferably, at least 2500 sensing electrodes.

[0066] As discussed above, the system of the present invention is

capable of displaying sufficient sensitivity to be able to respond to the
interaction of a single cell with a sensing electrode. This is true even for

electrodes with surface areas significantly larger than typical cells of interest.
However, as also mentioned above, the system of the present invention is
capable of a significant scaling up as represented by the simultaneous
monitoring of a large number of sensing electrodes interacting with cells from
one or more cell containment volumes. Although this scaling up contemplates
the use of typical sized sensing electrodes, it is also possible to utilize a two-
dimensional array of microelectrodes that can provide a unique, non-optical
picture of the movement of cells in response to various stimuli. In this manner,
the array of microelectrodes functions analogously to an array of pixels on a CRT
screen driven by a signal from a microprocessor. Instead of emitting light of
varying wavelengths in response to this signal, the microelectrode "pixels" of
this embodiment of the present invention provide an electrical signal that is
responsive to the interaction of individual cells with the electrode surface. By
coupling this system to the control of a microprocessor and computer software
executable on such microprocessor, it is possible to create a "picture" of
electrical signals generated from the array of microelectrodes that can be
displayed as an "image" of the motion of individual cells in the system. This
provides significant advantages in terms of sensitivity and in terms of monitoring
the complex factors controlling or affecting the movement of individual cells.

[0067] In certain of the examples provided below, Dictyostelium

discoideum cells have been used to study eukaryotic cell chemotactic
movement. Dictyostelium discoideum is an eukaryotic amoeba, which normally
inhabits the soil. During its life cycle, the haploid cells undergo two distinct
types of chemotactic movement. In the vegetative phase, the amoebae are
attracted to folic acid, which is released by their bacterial food source, and
detected by cell surface folate receptors. As the bacterial food source is
depleted, D. discoideum enters the developmental stage of its life cycle. The
number of folate receptors decreases during the first 7-9 hours of development
and the cells become responsive to cAMP released by other amoebae. The
number of cAMP receptors (cAR's) begins to increase immediately after the
initiation of starvation and cAR1 is maximally expressed on the surface 3-4
hours into development.

[0068] The cells of D. discoideum thrive at ambient conditions and their

mechanisms of motility are analogous to leukocytes. In moving across a

-22 -

substrate, these cells extend pseudopodia at their leading edge that attach to the
substrate and orient the cell in the direction of travel. Dictyostelium are known
to be chemotactic to a variety of agents. For example, folic acid produced by
bacteria establishes a gradient that allows vegetative Dictyostelium cells to find
their bacterial prey. Cyclic AMP (cAMP), a chemotactic signal produced by
Dictyostelium, is used during development to direct the aggregation of individual
cells to form the multicellular organism.

Examples
Example 1 Methods and Materials

[0069] Cells: Dictyostelium discoidium strain NC4A2 is an axenic cell line

derived from the wild type NC4 line. The myosin II heavy chain mutant (HK323)
was generated by homologous recombination to delete the coding portion of the
gene in the NC4A2 cell line. Cells were maintained in HL5 media in 100 mm
petri dishes with media changes every three days. Cells to be used for
experimental procedures were harvested at mid-log phase. Cells were
centrifuged at 200xg for 5 minutes at room temperature, re-suspended in fresh
media and counted with a Z2 particle counter (Coulter, Miami, FL). Chambers
were loaded with 10 6 cells per well.

[0070] Agarose preparation: Chemotaxis assays were adapted from the

under-agarose chemotaxis method. Briefly, a 0.5% solution of GTG agarose
(FMC Corporation, Rockland, ME) was prepared in 1x SM media (10g Difco
Bacto-Peptone, 10 g glucose, 1g yeast extract, 1 .9g KH 2 P0 4 , 0.6g K 2 HP0 4 ,
0.43g MgS0 4 per liter, pH 6.5). For chemokinetic assays, 2x SM agarose media
was premixed with an equal volume of 2X folic acid and then added to the
chamber to harden.

[0071] Under Agarose Chemotaxis Assay In order to prepare plates for

the under agarose assay, SeaKem GTG agarose (FMC BioProducts, Rockland,
ME) was melted at concentrations as indicated in SM medium (10g Difco Bacto-
Peptone, 10g glucose, 1g yeast extract, 1 .9g KH2 P04 , 0.6g K2 HP04 , and
0.43g MgS04 to 1L pH6.5) (23). Similar results were obtained with other types
of agarose (ultraPURE LMP (BRL), NuSieve GTG (FMC, Inc), Ultra Low Gelling

(FisherBiotech), and ultraPure (Gibco BRL)). Motility was generally higher when
SM was used to prepare the agarose instead of HL-5. The agarose mixture was
prepared fresh each day by mixing sterile SM (previously autoclaved) with the
agarose powder and autoclaving for 5 minutes, slow exhausting and then plating
as soon as possible. Four ml_ of agarose solution was added to each 60-mm
plastic petri dish and allowed to harden for 1 hour.

[0072] Reagents: The chemoattractant folic acid (Research Organics, Inc.

Cleveland, Ohio) stock at 100 mM was prepared by dissolving 0.44g folate in
220ul of 10 M NaOH. The final volume was adjusted to 10 ml with distilled
water. The solution was filter sterilized through a 0.2 micron filter, aliquoted and
stored frozen at -20°C in the dark. The folate solution was adjusted to the
appropriate concentration and added to wells one hour prior to the addition of
cells to allow for establishment of the chemotactic gradient. Cisplatin (Sigma
Chemical Co., St Louis, MO) was dissolved in phosphate buffered saline (PBS;
NaCI 8g, KCI 0.2g, KH 2 P0 4 0.2g Na 2 HP0 4 1.1 5g in 1000 ml distilled H 2 0) and
cells were incubated with three different concentrations of cisplatin in PDF (20
mM KCI, 5 mM MgCI 2 , 6H 2 0, 20mMKPO 4 , 0.5% dihydrostreptomycin sulfate,
pH 6.4) for one hour. Following this incubation, the cells were washed three
times in PDF and then re-suspended in PDF before placement in the cell wells of
EClS/taxis chambers.

[0073] Analysis system: The commercially available ECIS electrode

configuration (Applied Biophysics, Inc., Troy, NY) consists of 8 chambers per
array, each with a large electrode and a small target electrode (see panel A of
FIG. 2). These chambers were filled with 300 /jL of a 0.5% solution of melted
agarose prepared as described above. The thickness of the agarose layer is 4
mm. After the agarose had solidified, a sharpened 14 gauge cannula (Becton
Dickinson, Rutherford, NJ) was used to punch wells at appropriate locations in
the agarose. For chemotaxis assays, wells were located 2 mm on either side of
the target electrode along a common axis (see panel B of FIG. 2). The chambers
were chilled at 4°C for 15 minutes and then the agarose plugs were removed by
aspiration using a Pasteur pipette.

[0074] The chemoattractant was then loaded into one well in appropriate

chambers and the gradient was allowed to form for one hour. Cells were then
loaded into the other well and the apparatus was attached to the ECIS

-24-

instrumentation and the measurement of resistance was initiated. A current
flow of 1 volt a.c. at 4,000 Hz was passed through the chamber at 60 second
intervals. Impedance to this current flow was measured and a resistance value
was calculated according to established protocols. Resistance values could be
observed in real time on the computer display. In the results presented below,
the data is presented as normalized resistance, which is calculated as a fraction
of the initial resistance of the chamber at the start of the experiment.

[0075] Imaging: The gold electrode and overlying photoresist is thin

enough to be visually transparent. For some experiments, the area of the small
electrode was imaged during the collection of resistance data to establish the
time of arrival of cells. The plate was placed on the stage of a Leica DM IL
Microscope and images were captured every minute using a Dage CCD300 video
camera and a Scion CG-7 frame grabber. Image capture was controlled and
images processed using Scion Image software (Scion Inc., a derivative of NIH
Image developed by Wayne Rasband at NIH). In other experiments, the
chambers of a plate were periodically imaged in order to verify the data obtained
by ECIS measurements.

Example 2 Measurements of cells responding
to a simple chemotactic gradient.

[0076] A gradient was established by loading 1mM folate into the

chemoattractant well of a modified ECIS chamber, and one hour later

Dictyostelium cells were placed in the adjacent cell well. As a control, cells

were added in parallel to a well in a chamber in which no folate was added to

the chemoattractant well. In order to visualize the arrival of the cells at the

target electrode, the cells exposed to folate were continuously monitored by

video microscopy. During the time before arrival of cells at the electrode, both

the control chamber and the folate chamber show a continuous, smooth

decrease in resistance (labeled * 1 in FIG. 4). In the video images collected from

this electrode, the leading cells can be seen approaching the target electrode

from the lower left (FIG. 5, panel A). The first small peak at 3.75 hours (labeled

*2 in FIG. 5, panel A) coincides with the initial cell leaving the photoresist

surface and spreading on the target electrode (FIG. 5, panel B). As this first cell

crawls over the electrode the resistance remains above background, and then

decreases as that first cell moves off the electrode. Analysis of the time-lapse
data indicates that the cell does not alter its motile behavior significantly as it
changes from moving on the photoresist substrate to moving on the elemental
gold electrode surface. The resistance increases again (labeled *3 in FIG. 4) as
a wave of cells arrives at the electrode (FIG. 5, panel C). As the wave of cells
passes, the resistance begins to gradually decrease (FIG. 5, panel D).
Throughout the period of measurement, there is a correlation between the
number of cells on the electrode and the measured resistance values (FIG. 6).

[0077) The average speed of cell movement to the target electrode was

calculated to be approximately 10 /vm/min. This speed is consistent with
previous measurements of wild type Dictyostelium chemotaxis on an agar
surface (6 //m/minute± 1.2). In the assay system of the present invention, this
movement occurs primarily on a layer of photoresist material until the cells
eventually arrive at the gold target electrode surface. Cell movement is
unimpeded by the 5 micron step that the cells must traverse to reach the surface
of the gold electrode and must cross again as they depart the electrode at the
other side (data not shown). It is interesting to note that the resistance does not
change until the cell has actually contacted the electrode surface, and is
unchanged as the cell remains on the edge of the photoresist/electrode interface.

[0078] The normalized resistance changes over the period before cells

arrive at the surface of the target electrode in a way that appears to reflect
gradual changes to the electrolyte characteristics of the culture media. This
change probably reflects equilibration of the media with the external atmosphere,
combined with the impact of metabolic activity of the cultured cells on the
media. Since resistance is normalized to the initial reading within each chamber,
this shift does not interfere with the ability to note the increase in resistance
that attends the arrival of cells at the target electrode. Furthermore, the
changes in resistance that attend changes to the culture conditions over time are
not subject to the rapid transient changes in resistance that are characteristic of
cellular activity on the electrode.

#

26-

Example 3 Dictyostelium responds to folic acid in a dose dependent manner.
[0079] In order to assess the sensitivity of this technique to the

measurement of a range of chemotactic gradients, gradients were established by
adding a range of folate concentrations (from 0.16 to 1 mM) to the
chemoattractant well. Cells exposed to control media in the absence of
chemoattractant did not arrive at the target electrode during the course of the
experiment, hence no significant change to normalized resistance was observed
(data not shown). In contrast, cells placed in the gradient formed by 1 mM
folate were found to arrive at the target electrode at approximately 3.5 hours
after the placement of cells in the cell well. This movement was considerably
faster than the response of cells to the gradients established by either 0.5 or
0.16 mM folate. In these instances, cell arrival at the target electrode began at
about 5 to 5.25 hours after addition of cells to the system. See FIG. 7.
[0080] Another notable difference that distinguishes the cells responding

to each level of chemoattractant is that the absolute number of cells that arrive
decreases as the dose of folate used to establish the gradient decreases. This is
presumably a consequence of cells that are exposed to a suboptimal
concentration of folate. One important advantage of the system of the present
invention over other prior art techniques is that it allows the assessment of the
whole cell population during the migratory process as opposed to looking at the
fastest cells among the population. By analyzing the entire data set one can
estimate the number of cells that have responded to the signal and the duration
of time over which cells continue to arrive at the electrode. The observation
that resistance increases in a manner proportional to the number of cells on the
target electrode suggests that individual resistance values can be interpreted to
reveal the size of the responding cell population. When testing new chemokines
or inhibitors, this data will provide more information about the response of the
population than a single time point assay.

Example 4 EClS/taxis measurements in a uniform concentration of folate.
[0081] Another way to configure a chemotaxis assay is to add the

chemoattractant uniformly throughout the agarose matrix. If the cells affect the
chemoattractant (either by consuming it or by secreting enzymes that degrade

it), a local gradient will form and cells will then move toward areas of higher
chemoattractant concentration. For example, Dictyostelium secretes folate
deaminase, which can destroy nearby folate and thereby create a folate gradient.
Cells can also respond to some agents by increasing their speed of random
movement (chemokinesis) which can also result in accelerated movement away
from the origin.

[0082] In the data illustrated here, folate was mixed with the agarose

before it was poured in the chamber, so that it was present at a uniform
concentration throughout the chamber and surrounding the cell well. When the
agarose contained 0.5 mM folate, the time of arrival of cells at the electrode
was similar to arrival times for chemotactic responses to lower concentrations of
folate, as illustrated in FIG. 8. Intriguingly, the cells arrive at the electrode as a
wave rather than as a continuous stream even though there is a large reservoir
of cells that remain in the cell well. This may result from cells altering the local
concentration of folate, thus limiting the movement of cells behind the initial
wave. It was interesting to note that a very low concentration of folate (0.013
mM folate) can accelerate the movement of a smaller number of Dictyostelium
to a greater degree than higher concentrations. When cells are exposed to
0.013 mM folate concentration, they arrive at the target electrode approximately
1 to 1 .25 hours earlier than they do when exposed to 0.04 or 0.5mM folate.

Example 5 Identification of cell lines that are unable
to produce a chemotactic response.

[0083] An additional utility of the system of the present invention is in the

identification of new mutations that contribute to the chemotactic and
chemokinetic processes. In a prototypical experiment, a myosin II mutant was
used that has previously been shown to have a reduced ability to respond to
cyclic AMP and which is unable to move normally during morphogeneis. The
response of these cells to a folate gradient has not been previously reported.
The myosin II mutant cells did not arrive at the target electrode at any point
during the course of the experiment (FIG. 9). Preliminary analysis has shown
that at these cells do not move sufficiently far under agarose to reach the target
electrode. Manipulation of the gel overlay composition may allow other aspects
of cellular behavior to be examined with this technique.

Example 6 : Dose-dependent inhibition of chemotactic responses by cisplatin.

[0084] A significant potential use of the system of the present invention

is in the identification of pharmacological inhibitors of chemotaxis or
chemokinesis. Previous work has shown that cisplatin can decrease the
chemotactic responses of Dictyostelium. When this phenomenon was examined
in the system of the present invention, the time of arrival of cells at the
electrode was delayed in a manner proportional to the dose of cisplatin (FIG.
10). While untreated cells reached the electrode after 4.4 hours, treated cells
arrived at times that extended from 5.6 to 8 hours after the start of the
experiment. The drug did not have a discernable effect on cell viability at the
concentrations used, since it did not appear to affect the number of cells that
eventually arrived at the electrode. Cisplatin may inhibit cell movement, through
inhibition of association of actin with the cortex and/or via interactions with the
signal transduction cascade. This result illustrates the potential of the system of
the present invention for high throughput screening of potential agonists and
antagonists of chemotactic behavior.

Example 7 Chemotactic Response of Bone Marrow Cells
As Measured in a Three-Electrode System

[0085] Bone marrow cells from BALB/c mouse were placed in the cell well

of the apparatus according to procedures described above. Guinea pig serum
(diluted 50:50) was simultaneously added to a chemoattractant well. The cell
assay system was as described above with the addition of a reference electrode.
With this three-electrode configuration, measured changes in resistance
attributed to the arrival of cells at the sensing electrode can be corrected for
changes in background resistance unassociated with cell interaction with the
surface of the sensing electrode. Thus, measured resistance values at the
sensing electrode that result from the conductive behavior of the media were
subtracted from the total resistance values measured at the sensing electrode by
subtracting the reference electrode readings from the sensing electrode readings
at each time point. Resistance data as presented in FIG. 1 1 is presented from
the initial time point one hour after beginning of culture. Each of the numbered

points on the resistance plot of FIG. 1 1 represents and individual still photograph
taken at that time point. The corresponding still photos are provided in FIG. 12,
panels A through D.